purpose. The chemical coding of intrinsic choroidal neurons (ICNs) has features in common with extrinsic fibers (e.g., from the pterygopalatine ganglion) making it impossible to assess whether a neuronal nitric oxide synthase (nNOS)/vasoactive intestinal polypeptide (VIP)–immunoreactive nerve fiber is of intrinsic or extrinsic origin. Neurobiotin injections into single neurons allow the visualization of projections of these cells and the determination of the origin of target innervation. Thus, this technique was used in the present study to help characterize the organization of the ICN in the human eye.

results. ICN processes were traced over distances of up to 2.612 μm. They were found in the immediate vicinity of other nNOS-positive or -negative ICNs and were also found apposed to smooth muscle fibers (vascular and stromal nonvascular). CGRP-positive fibers forming boutons were observed closely associated with ICNs. Electrophysiological recording showed phasic firing without slow afterhyperpolarization, no spontaneous activity, an input resistance of 136 ±73 MΩ, and a membrane time constant of 7 ± 1 ms.

conclusions. Apart from the first functional characterization of ICNs, this study provided more precise evidence of reciprocal ICN-to-ICN contacts and innervation of both choroidal nonvascular and vascular smooth muscle. The presented technique offers promising perspectives to further investigate the function of ICNs in ocular homeostasis.

The autonomic innervation of the vertebrate eye is mediated through sympathetic and parasympathetic pathways originating in superior cervical, ciliary, and pterygopalatine ganglia, respectively. As a third component, trigeminal primary afferent neurons release active substances from their peripheral terminals in the choroid.12 This classic scheme does not take into account the presence of autonomic nerve cells residing within the choroid of the eye, the intrinsic choroidal neurons (ICNs). Though first described some 150 years ago,3 they remained poorly recognized despite repeated reports.456 These neurons received renewed interest when it was found that they contained vasoactive intestinal polypeptide (VIP)7 and neuronal nitric oxide synthase (nNOS)89 and when they were commonly observed in reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)–stained choroidal wholemounts in both higher primates8 and birds.10

Putative targets that might be innervated by ICNs include arteries and nonvascular smooth muscle fibers of the choroid.891112 Thus, ICN may be involved in the control of ocular blood flow, choroidal thickness, and intraocular pressure. In primate eyes, no clear-cut marker substance of ICN is known. Nerve terminals on ICN costaining for VIP and nNOS may be of either intrinsic or extrinsic origin, such as from pterygopalatine ganglion cells, because in the latter neurons exhibit a neurochemical coding similar to that of ICNs.1314

The purposes of the present study were to develop a method for intracellular recording and dye filling of single ICNs in the human choroid and to perform a basic morphologic and electrophysiological characterization of the neurons identified by this method.

Materials and Methods

Tissue Preparation

In this study, 15 choroids of human donors (65–89 years of age; both sexes) were investigated, according to the Declaration of Helsinki for the use of human tissue in research. Donor bulbi were obtained from the cornea bank of the University of Antwerp (UIA), Belgium, within 4 to 12 hours after death. Eyes were cut open circumferentially around the ora serrata, and retina and vitreous body were removed. Residual scleral cups with the choroid attached were gently rinsed in a standard ice-cooled oxygenated (95% O2, 5% CO2) Krebs-Ringer solution of the following composition (in mM): 118.0 NaCl, 4.75 KCl, 2.54 CaCl2, 1.2 MgSO4, 1.0 NaH2PO4, 25.0 NaHCO3, and 11.1 glucose. Retinal pigment epithelium was detached carefully using cotton-wool tips. Choroids were dissected and transferred into ice-cooled oxygenated Krebs-Ringer solution. Before the electrophysiological experiments began, the choroids were pinned flat in plastic dishes coated with a silicone elastomer (Sylgard; Dow-Corning Europe, La Hulpe, Belgium).

Fixation of the Tissue with Low-Gelling-Temperature Agarose

Intracellular impalements of human ICNs were hampered by loose fibers of the lamina suprachoroidea floating on top of the preparation. Fixation of these loose fibers was achieved by covering the preparation with a thin layer of agarose, using the following protocol. First, a stock solution of 1.6% agarose (SeaPlaque; Duchefa, Haarlem, The Netherlands) was made and kept at a temperature of more than 40°C. This stock solution was mixed with Krebs-Ringer solution (1:1) and heated up to 60°C for several minutes. The Krebs-agarose mixture was then cooled down to 40°C to 50°C, and a thin layer was put on top of the preparation. To allow the agarose to gel, the preparations were incubated for another 30 minutes at 4°C. Once the agarose had completely gelled, the recording dish was transferred into ice-cooled oxygenated Krebs-Ringer solution.

Visualization of ICNs

Therefore, a 4 μM solution of the fluorescent vital dye 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-ASP; Molecular Probes, Eugene, OR) in oxygenated Krebs-Ringer solution was used (20 minutes at 37°C). The validity of this vital staining method has been demonstrated in different tissues,1516 including human choroid.17 During the staining procedure and afterward, the specimens were shielded from light and stored in ice-cooled oxygenated Krebs-Ringer solution.

Measurements were made after allowing the impalement to stabilize for a few minutes (until the resting potential of the cells stabilized) without applying intracellular current. To investigate passive and active membrane properties, current step commands were created on computer (pClamp ver. 6.0.2; Axon Instruments). The data were low-pass filtered online (3 kHz), digitized (sample frequency 5 kHz), and stored. The input resistance and membrane time constant of the impaled neurons were estimated by passing small hyperpolarizing current pulses of variable amplitude (−0.05 to −0.3 nA) through the intracellular recording electrode. The resulting membrane potential change was measured so that current–voltage curves could be constructed. Each step was repeated three times, and the average value was used for further calculations. Durations of action potentials were measured as half widths (i.e., the time interval between the point on the upstroke at which the amplitude of the action potential is halfway between the membrane resting potential and the maximum potential, and the equivalent point on the downstroke). To avoid interference of voltage changes due to the depolarizing current applied, short pulses of 2 ms were used to evoke a single action potential, whenever individual action potential characteristics were measured. Data analysis was performed on computer (pClamp, ver. 6.0.2; Axon Instruments, Excel 97; Microsoft, Redmond, WA; and SigmaPlot, ver. 5.0; SSPS Sciences, Chicago, IL). All data are presented as the mean ± SD.

Immunohistochemistry

During electrophysiological recordings, impaled neurons were iontophoretically filled with neurobiotin by passing depolarizing current pulses (0.5–1 nA, 100–500 ms duration) through the recording electrode. After being electrophysiologically recorded, the impaled cell was photographed and its position in the choroid, mainly in the temporal quadrants, was mapped for later localization and morphologic identification. The preparations were eventually fixed in a modified Zamboni solution (4% paraformaldehyde, 0.2% picric acid, and 0.1 M sodium phosphate buffer) for 2 hours at room temperature. After fixation, the preparations were further processed to improve conditions for immunocytochemistry. To this end, they were first rinsed in phosphate-buffered saline (PBS; pH 7.4, 0.01 M) for 10 minutes, followed by a rinse in 50% ethanol (5 × 8 minutes) and rehydrated in PBS (5 × 8 minutes). The preparations were then incubated in 0.05% thimerosal (Sigma-Aldrich) for 30 minutes and rinsed again in PBS (3 × 10 minutes). To enhance antibody penetration and to prevent nonspecific binding of antibodies, the preparations were incubated in PBS (30 minutes at room temperature) containing 10% normal goat serum (Dako, Glostrup, Denmark) or normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA), depending on the secondary antibody used, and 0.1% bovine serum albumin, 0.05% thimerosal, 0.01% NaN3, and 1% Triton X- 100 (all Sigma-Aldrich). The preparations were then incubated (36 hours; room temperature) for triple labeling in primary antisera and antibodies against nNOS (1:1000, raised in rabbit; EuroDiagnostica, Malmö, Sweden) and α-smooth muscle actin (clone1A4, 1:2000, mouse; Sigma-Aldrich, or nNOS and human calcitonin gene-related peptide [CGRP], 1:1000, guinea-pig; Euro-Diagnostica), respectively. After six washes (5 minutes each) in PBS, the preparations were incubated in secondary antisera for 6 hours at room temperature in corresponding fluorescein isothiocyanate (FITC; for nNOS; 1:100) or Cy5 (for SMA and CGRP, 1:200; both from Jackson ImmunoResearch Laboratories) tagged antibodies. To reveal the presence of neurobiotin in the impaled neurons, the tissue was incubated with streptavidin coupled to Cy3 (1:2000; Jackson ImmunoResearch Laboratories). After a final wash to remove the unbound secondary antibodies, the preparations were mounted in antifade medium (Vectashield; Vector Laboratories).

From the group of 15 choroids in this study, 9 neurons were successfully impaled. Five of them appeared to be immunoreactive for nNOS.

All impaled neurons showed a clear labeling for neurobiotin in both cell soma and processes. Largest cell body diameter of these group of neurons, as measured in projected confocal Z-stacks, ranged from 19 to 35 μm. Confocal images showed eight cells with a clearly discernible, eccentrically positioned nucleus. Cell bodies were ovoid or droplike in shape and mainly had a smooth contour with excavations of the cell body presumably caused by surrounding satellite cells. These neurons gave rise to up to seven processes: up to four short processes less than 60 μm in length and one to three long processes up to 2.612 μm (Fig. 1G) . Processes arose either radially or polarly from the impaled cell bodies. Only in one case was a process visualized in the confocal microscope thought to be an axon based on the presence of a hillocklike appearance (Figs. 1F2B) . The projection of this axon-like process was observed over a distance of 887.7 μm, giving rise to a short (64.7 μm; 215 μm off the cell body) and a long (357.7 μm; 341.6 μm off the cell body) collateral. A detailed analysis of the length of these processes, however, did not allow a further subclassification of these neurons (see Table 1 ).

Neurobiotin-filled varicose fibers forming boutons could be followed over long distances, sometimes crossing their own pathways in wide turns (Figs. 1B1G) and running parallel to choroidal blood vessels (Fig. 2A) . Detailed analysis of confocal Z-series, using antibodies against α-smooth muscle actin, revealed neurobiotin-positive fibers in close apposition to the vessel wall, sometimes coursing along the media (Figs. 3A3B3C3D) .

Neurobiotin-filled fibers were found to run parallel to nonvascular smooth muscle fibers of the choroid (Fig. 3E3F3G) , forming bouton-like approaches. Neurobiotin-filled processes of impaled cells showed boutons and closely embracing nerve fibers on other nitrergic (Fig. 4A) and nonnitrergic cells in the same ganglion, or with neurons in ganglia at a certain distance (up to 800 μm; Fig. 4B ). In contrast, close appositions of intrinsic nitrergic neurons onto neurobiotin-filled ICNs up to 400 μm removed from the impaled cells were also detected (Figs. 4C4D) . A neurobiotin-filled ICN immunonegative for nNOS is depicted in Figures 4Eand 4E ′. Neurobiotin-positive fibers forming varicosities projected along with nNOS-immunoreactive fibers in the same nerve bundles (Fig. 4F) .

CGRP-immunoreactive fibers coursed through the choroid, forming varicose fibers with small boutons. These fibers were found to run parallel to nNOS-immunoreactive fibers (Fig. 4G) , presumably originating from ICNs, but an extrinsic origin cannot be excluded. Small CGRP-immunoreactive boutons were closely attached to cell bodies of both impaled and nonimpaled nNOS-negative and nNOS-positive ICNs (Fig. 4H) . Close appositions of CGRP-positive boutons on processes of nNOS-positive ICNs were also observed (Fig. 4I) .

Electrophysiology

Approximately 30% of the impaled neurons of the human choroid gave stable electrophysiological recordings (resting membrane potential: −52 ± 10 mV) and fired action potentials after direct somal depolarization, with a clear reversal (“overshoot”) of the resting membrane potential (Fig. 5A) . The neuron shown in Figures 5Eand 5E ′ had a stable membrane resting potential (−50 mV) and an input resistance of 61 MΩ but was not able to fire action potentials after direct somal depolarization, not even during long-lasting (200–500 ms) depolarizing current pulses. None of the impaled neurons showed spontaneous activity.

The neurons that fired action potentials all displayed rather brief spikes (action potential half-width: 1.6 ± 0.6 ms) with a monophasic repolarization, which is reflected in the first-time derivative of the voltage trace without inflections (Figs. 5B5b ′). During the application of long-lasting (200–500 ms) suprathreshold depolarizing current pulses, the neurons fired action potentials only at the onset of the depolarization, irrespective of the applied current strength (Fig. 5C) , which is indicative of phasic firing behavior. The action potentials were not followed by a slow afterhyperpolarization (Fig. 5D) .

The mean input resistance of the neurons was 136 ± 73 MΩ, and the mean membrane time constant of these neurons amounted to 7 ± 1 ms.

Discussion

This study presents for the first time electrophysiological recordings from intrinsic neurons of the human choroid. They show phasic firing behavior but no spontaneous activity. Iontophoretic filling of impaled ICNs using neurobiotin revealed single nerve cells and their projections over distances as long as 2.612 μm. Two additional procedures were crucial for successful impalements with microelectrodes: (1) the use of the low-gelling-temperature agarose covering the tissue immobilized the neurons and prevented the loose connective tissue fibers from adhering to the microelectrode, and (2) the application of the supravital 4-Di-2-ASP staining enabled the microscopic visualization of the ICNs. This dye has no influence on basic electrophysiological neuronal features.15161819 Although agarose has been used earlier to prevent single neurons or tissue slices from moving in the recording chamber,20212223 we used it, to our knowledge, for the first time on vital wholemount preparations followed by immunohistochemistry. Comparison of the staining technique using the vital fluorescent dye 4-Di-2-ASP in agarose-treated versus nontreated tissue yielded no differences. In addition, the penetration of antibodies used in our immunohistochemical protocols was not hampered by the agarose technique, and, as reported earlier,2023 no interference of agarose with electrophysiological recordings was observed. Therefore, this technique appears to be a valid tool for future pharmacofunctional studies of ICNs. Nevertheless, the intracellular recording technique used in this study can cause a leak current at the pipette–membrane interface and can cause cell damage, either of which could result in depolarization. Future studies involving various configurations of a patch–clamp approach may be helpful to the characterization of the electrophysiology of these neurons.

Donor eyes available in this study were those of elderly persons; hence, age-related alterations of the tissue cannot be ruled out.242526 However, one advantage pertains to the reduced choroidal pigmentation observed in elderly compared with younger choroids, rendering identification of the supravitally stained ICNs easier in the inverted microscope setup.

Application of neurobiotin as an intracellular morphologic marker revealed a rather “simple” morphology of human ICNs, showing neurons with a bi- or multipolar appearance. This observation is in accordance with earlier studies using neurochemical markers, such asVIP, nNOS or NADPHd.789 Recently, antibodies against different neurofilament subunits have been used in a study of ICNs.27 Iontophoretic filling, however, has the advantage that the intrinsic origin of the neurobiotin-positive fibers can unequivocally be determined, even when the neurochemical features of intrinsic and extrinsic fibers overlap, as is the case in the human choroid. Apart from the ICNs, the parasympathetic postganglionic neurons of the pterygopalatine ganglion supplying the eye228 also contain VIP and nNOS/NADPHd.1314 Other extrinsic fibers in the human choroid contain only one or even none of these two markers: no VIP-positive neurons were detected in the ciliary ganglion,14 whereas in the superior cervical ganglion, few neurons show immunoreactivity for VIP, but not for nNOS.29 In the trigeminal ganglion, a minority of the cells is positive for nNOS, but colocalize CGRP.30

Contacts of ICNs to vascular smooth muscle cells have been suggested in earlier studies,89 but direct evidence has not been provided yet. By contacting vascular smooth muscle cells, as our results show, ICN may influence the muscle tone of choroidal blood vessels and thus be considered an important component in the autonomic regulation of choroidal blood flow; thus, they may play an important role in the autonomic regulation of intraocular pressure.313233 The function of the other nonneuronal target of ICNs (i.e., stromal nonvascular smooth muscle cells) is less obvious. Nonvascular smooth muscle cells were described in the choroid of humans,31134 other mammalians,113536 and various avian species.12 In humans, they are mainly concentrated in the temporal and submacular regions of the choroid.1134 Of note, these are the regions where ICNs also accumulate.82737 Influencing the contractile state of these nonvascular smooth muscle fibers may result in a change in the thickness of the choroid, leading to a shift of the adjacent retina and thus altering the optic properties of the eye. However, whether this is also the case in human eyes still must be elucidated. This mechanism has been extensively investigated in the avian eye3839 but the underlying mechanisms are poorly understood. ICNs may be suitable candidates for the neuronal control of these refractive changes.124041 Possible targets of ICNs other than those expressing smooth muscle actin are still to be determined.

The observed close appositions of CGRP-immunoreactive fibers, most likely of trigeminal primary afferent origin, with ICNs may represent a precentral reflex arrangement similar to the spinal visceral afferent contacts on neurons in prevertebral ganglia.42 Contacts of CGRP-positive nerve fibers were not described in the human pterygopalatine ganglion, nor were CGRP1 receptors expressed.43 In the avian eye, CGRP-positive contacts on ICNs have been recently reported, presumably forming the anatomic basis of a precentral reflex arc.40 Therefore, it is reasonable to assume that CGRP-positive fibers in close apposition to human ICNs are also integrated in a precentral reflex arc. Our experiments with neurobiotin injections revealed that nerve fibers of intrinsic origin closely appose other ICNs. Although the appositions of both intrinsic and extrinsic nerve fibers on ICNs are suggestive of synaptic contacts, this has to be verified at the ultrastructural level. Further electrophysiological recordings are necessary to determine whether different functional types of ICNs are present in the human eye. Nevertheless, the technique developed herein enables functional studies of human ICNs in vitro, which will yield a better insight into the mechanisms regulating ocular homeostasis.

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